Yang et al. Journal of Experimental & Clinical Cancer Research 2014, 33:14 http://www.jeccr.com/content/33/1/14

RESEARCH

Open Access

Clinical implications of high NQO1 expression in breast cancers Yang Yang1†, Yan Zhang2†, Qunying Wu3, Xuelian Cui1, Zhenhua Lin1, Shuangping Liu1* and Liyan Chen1,4*

Abstract Background: NAD (P) H: quinone oxidoreductase 1 (NQO1) is a xenobiotic metabolizing enzyme that detoxifies chemical stressors and antioxidants, providing cytoprotection in normal tissues. However, high-level expression of NQO1 has been correlated with numerous human malignancies, suggesting a role in carcinogenesis and tumor progression. This study aimed to explore the clinicopathological significance of NQO1 and as a prognostic determinant in breast cancer. Methods: A total of 176 breast cancer patients with strict follow-up, 45 ductal carcinoma in situ (DCIS), 22 hyperplasia and 52 adjacent non-tumor breast tissues were selected for immunohistochemical staining of NQO1 protein. Immunofluorescence staining was also performed to detect the subcellular localization of NQO1 protein in MCF-7 breast cancer cells. Eight fresh breast cancers paired with adjacent non-tumor tissues were quantified using real time RT-PCR (qRT-PCR) and western blot. The correlations between NQO1 overexpression and the clinical features of breast cancer were evaluated using chi-square test and Fisher’s exact tests. The survival rate was calculated using the Kaplan–Meier method, and the relationship between prognostic factors and patient survival was also analyzed by the Cox proportional hazards models. Results: NQO1 protein showed a mainly cytoplasmic staining pattern in breast cancer. The strongly positive rate of NQO1 protein was 61.9% (109/176) in breast cancer, and was significantly higher than in DCIS (31.1%, 14/45), hyperplasia tissues (13.6%, 3/22) and adjacent non-tumor tissues (13.5%, 7/52). High-level expression of NQO1 protein was correlated with late clinical stage, poor differentiation, lymph node metastasis, Her2 expression and disease-free and 10-year overall survival rates in breast cancer. Moreover, multivariate analysis suggested that NQO1 emerged as a significant independent prognostic factor along with clinical stage and Her2 expression status in patients with breast cancer. Conclusions: High-level expression of NQO1 appears to be associated with breast cancer progression, and may be a potential biomarker for poor prognostic evaluation of breast cancers. Keywords: Breast cancer, NQO1, Immunohistochemistry, Prognosis

Background Breast cancer is one of the most common malignancies in women worldwide and the second leading cause of cancer death among women [1,2]. Studies over the past several decades have found that the expression profiles of estrogen receptor (ER), progesterone receptor (PR) * Correspondence: [email protected]; [email protected] † Equal contributors 1 Department of Pathology & Cancer Research Center, Yanbian University Medical College, Yanji 133002, China 4 Department of Biochemistry, Yanbian University Medical College, Yanji 133002, China Full list of author information is available at the end of the article

and human epidermal growth factor receptor 2 (Her2)/ neu are closely related with breast cancer, and have been used for predicting the outcome and response to breast cancer therapy [3,4]. Although numerous advances in prevention, surgical resection, and adjuvant chemoradiotherapy have led to a decline in the overall mortality due to breast cancer, the survival rates for patients with metastatic disease have not significantly improved [5,6]. Consequently, the discovery of novel biomarkers involved in the diagnosis and progression of breast cancer is of great value.

© 2014 Yang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Yang et al. Journal of Experimental & Clinical Cancer Research 2014, 33:14 http://www.jeccr.com/content/33/1/14

NAD (P) H: quinone oxidoreductase 1 (NQO1), also known as DT-diaphorase, menadione reductase, or quinone reductase 1, is a cytoplasmic flavoenzyme encoded by a gene located on chromosome 16q22. NQO1 uses NADH or NADPH as substrates to directly reduce quinones to hydroquinones [7,8]. Functions of NQO1 include xenobiotic detoxification, superoxide scavenging and the maintenance of endogenous antioxidant vitamins [9]. It is conceivable that NQO1 plays an important role in protecting normal cells against oxidative injury and carcinogenesis. Paradoxically, despite the cellular functions of this “cell protector”, the antioxidant role of NQO1 was suggested by evidence that the disruption of the NQO1 gene or genetic polymorphism increased the risk of chemical-induced toxicity and cancers [10,11]. NQO1 has been found to be expressed at high levels in many human tumors, including breast cancer, melanoma, lung cancer, cholangiocarcinoma and pancreatic cancer [12-15]. In addition, the high level of NQO1 expression in solid tumors in combination with the ability to reduce many quinine-containing antitumor drugs has dawn attention to NQO1 as a potential molecular target in cancer treatment [16,17]. However, the clinical significance of NQO1 expression status in breast cancer remains unclear. In this study, we demonstrated the clinicopathological significance of NQO1 through prognostic evaluation of NQO1 overexpression in breast cancers. The results revealed that NQO1 protein is frequently upregulated in breast cancers compared with hyperplasia and adjacent non-tumor breast tissues. These findings indicate that NQO1 may be a good independent predictor of prognosis for patients with breast cancer.

Materials and methods Ethics statement

This research complied with the Helsinki Declaration and was approved by the Human Ethics Committee and the Research Ethics Committee of Yanbian University Medical College. Patients were informed that the resected specimens were stored by the hospital and potentially used for scientific research, and that their privacy would be maintained. Follow-up survival data were collected retrospectively through medical record analyses. Clinical samples

Eight fresh breast cancers paired with adjacent nontumor tissues were snapfrozen in liquid nitrogen and stored at −80°C until use. The histopathology of each specimen was reviewed on the hematoxylin and eosinstained tissue section to confirm diagnosis and tumor content at least 70% of tumor cells in the tissue sample. The study of 176 paraffin embedded breast cancer samples, as well as 45 ductal carcinoma in situ (DCIS)

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samples, 22 hyperplasis and 52 adjacent non-tumor tissues were also conducted. These samples were selected randomly from patients who underwent surgery between 2002 and 2009, with strict follow-up for survival status. Clinicopathological classification and staging were determined according to the American Joint Committee on Cancer (AJCC) criteria. Clinical information of the samples is summarized in Table 1. Immunohistochemical (IHC) analysis

IHC analysis was performed using the DAKO LSAB kit (DAKO A/S, Copenhagen, Denmark). Briefly, to eliminate endogenous peroxidase activity, 4 μm thick tissue sections were deparaffinized, rehydrated and incubated with 3% H2O2 in methanol for 15 min at room temperature (RT). The antigen was retrieved at 95°C for 20 min by placing the slides in 0.01 M sodium citrate buffer (pH 6.0). The slides were then incubated with the NQO1 monoclonal antibody (1:200, A180: sc-32793, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After incubation with the biotinylated secondary antibody at RT for 30 min, the slides were incubated with a streptavidin-peroxidase complex at RT for 30 min. IHC staining was developed using 3,3′-diaminobenzidine, and Mayer’s hematoxylin was used for counterstaining. We used tonsil sections as the positive control and mouse IgG as an isotope control. In addition, tissue sections were processed omitting the primary antibody as the negative control. Two pathologists (Lin Z & Liu S) who did not possess knowledge of the clinical data examined and scored all tissue specimens. In case of discrepancies, a final score was established by reassessment by both pathologists on a double-headed microscope. Briefly, the IHC staining for NQO1 was semi-quantitatively scored as ‘–’ (negative) (no or less than 5% positive cells), ‘+’ (5–25% positive cells), ‘++’ (26–50% positive cells) and ‘+++’ (more than 50% positive cells). The cytoplasmic expression pattern was considered as positive staining. Tissue sections scored as ‘++’ and ‘+++’ were considered as strong positives (high level expression) of NQO1 protein. Immunofluorescence (IF) staining analysis

IF staining was used to detect the sub–cellular localization of NQO1 protein in MCF-7 breast cancer cells. All steps were performed at RT. MCF-7 cells were grown on coverslips to 70–80% confluence, then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100 for 10 min after 24 h. After blocking with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the NQO1 antibody (1:500) at 4°C overnight, and followed by incubation with Alexa Fluor® 568 goat anti-mouse IgG (H + L) (A11004,

Yang et al. Journal of Experimental & Clinical Cancer Research 2014, 33:14 http://www.jeccr.com/content/33/1/14

Table 1 Correlation between NQO1 protein expression and the clinicopathological parameters of breast cancer Variables

No. of cases

NQO1 strongly positive cases (%)

Age ≥50

94

61 (64.9%)

Clinical implications of high NQO1 expression in breast cancers.

NAD (P) H: quinone oxidoreductase 1 (NQO1) is a xenobiotic metabolizing enzyme that detoxifies chemical stressors and antioxidants, providing cytoprot...
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